This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long term goal of this proposal is to elucidate the protease signaling pathways involved in innate immunity. We propose to identify the proteins that are cleaved during the innate immune response, and the proteases responsible. Activation of proteases, notable the inflammatory caspases, is a hallmark of the innate immune response, and regulates responses to bacterial infection, gout, arthritis, and vaccinations. Surprisingly very few (less than 10) proteins have previously been identified as substrates of the inflammatory caspases. From our work and that of others, there are now known to be greater than 500 proteins cleaved by caspases during apoptosis (regulated cell death). The inflammatory and apoptotic caspases have very similar catalytic efficiencies and the substrate specificities of the inflammatory caspases do not appear more restricted. What accounts for the 50-fold difference in the apparent number of proteins known to be processed by these two families? We propose to identify additional inflammatory caspase substrates and identify their roles in inflammation. We have begun to address these questions using a new and general mass spectrometry-based method for global profiling of proteolysis in cells (we call "degradomics"). In collaboration with the Burlingame lab we have developed an MS-based method to identify greater than 200 new apoptotic caspase substrates. Preliminary proteomic experiments with the inflammatory caspases have identified numerous (greater than 30) pro-inflammatory substrates, several of which play known roles in inflammation. Quantitative mass-spectrometry will be used to expand this list and validate the identified substrates. Selected hits will be further confirmed via western blot and analyzed for functional significance using ELISA-based methods.